1. Field of the Invention
This invention relates to reagents, methods and apparatus for the isolation of cellular components such as ribonucleic acid (RNA) from natural cellular sources.
2. Description of the Related Art
Cells contain a wide variety of cellular components appropriate to their function. They contain, for example, DNA, ribonucleic acid (RNA) and their expression products including a host of proteinaceous materials. This invention is useful for the isolation of such cellular components, but for convenience, the invention will be described principally as it applies to the isolation of RNA.
RNA is a critical component in the sequence of biological reactions which results in the expression of the myriads of proteins including hormones, enzymes and structural tissue essential for the existence of all forms of life. There is a critical need for large amounts of RNA for research purposes as well as diagnostic and therapeutic uses.
Plant cells and microorganisms including parasites, yeast and, bacteria, are potential sources of RNA, but because the cell wails of RNA sources are so strong, it is difficult, time consuming and may require expensive equipment to utilize them as RNA sources.
RNA isolation from bacteria is difficult because the cell walls are not readily susceptible to lysis. Current protocols for isolating RNA from bacteria frequently employ enzymes such as lysostaphin or lysozyme to lyse the bacterial cells followed by the addition of denaturing agents to inactivate the ribonucleases. However, these methods have not proven to be useful for the isolation of large amounts of high purity RNA.
U.S. Pat. No. 4,843,155 employs an RNA isolation procedure in which biological tissue such as mouse anterior pituitaries are initially homogenized in a "glass-teflon homogenizer" in an aqueous buffered medium at pH 4 containing phenol, a guanidinium salt such as guanidinium thiocyanate and, possibly an antioxidant. The homogenate is then extracted with an organic solvent insoluble in the buffer. Chloroform is exemplified as a useful solvent. The RNA remains dissolved in the aqueous buffer. The DNA and protein of the biological tissue concentrate at the interface between the organic and inorganic phases. The RNA is separated from the aqueous phase by precipitation with a water soluble alkanol such as isopropanol. The RNA may be recovered by centrifugation and removing of the supernatant. The patentee states that the isolation procedure can be completed in three hours.
This procedure is inconvenient because guanidine salts are expensive and malodorous. Moreover, as described, the method is limited to biological tissue (i.e., eukaryotic and not prokaryotic cells) as an RNA source and requires an extended period of time to complete. The cells in biological tissue, being eukaryotic, are enclosed by a fragile membrane which is easily ruptured.
Moreover, although glass beads have been used in methods of RNA isolation, glass beads cannot be used in conjunction with a homogenizer-type apparatus to achieve the results of the present invention, because beads would cause the homogenizer to jam. Glass beads have been used in conjunction with a bead beater to isolate nucleic acids from cells. However, the disadvantage to this method lies in the fact that the method smashes the cells, releasing the contents into medium in which unstable cellular components, such as RNA, are rapidly degraded by enzymes that are released.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of isolating cellular components such as RNA from cells, it should be apparent that there still exists a need in the art for reagents, methods and apparati suitable for the efficient isolation of such components from both eukaryotic and prokaryotic cells.